In 1975, Porath introduced immobilized metal ion affinity chromatography (IMAC) for fractionating proteins [J. Porath, J. Carlsson, I. Olsson, and G. Belfrage, Nature (London) 258, 598-599 (1975)]. In Porath's work, IMAC consists of derivatizing a resin with iminodiacetic acid (IDA) and chelating metal ions to the IDA-derivatized resin. Proteins bind to the metal ions through unoccupied coordination sites and are immobilized on the column. Since then, workers have used ligands other than IDA to chelate metal ions to resins. Studies with serum proteins have shown IMAC to be an extremely specific and selective separation technique [J. Porath and B. Olin, Biochemistry 22, 1621-1630 (1983)].
It is recognized that certain amino acid residues such as histidine, cysteine, methionine, glutamic acid, aspartic acid, lysine, and tyrosine, present in metalloprotein active sites, are responsible, at least in part, for the actual binding of free metal ions to such apoproteins. The actual mechanisms which give rise to the binding of proteins to free metal ions are not well understood and are dependent upon a number of factors, not the least of which is the conformation of the particular protein. However, when the metal ions are immobilized, at least three additional limiting factors come into play, viz., reduced number of available coordination sites on the metal, restricted accessibility of the tethered metal to the binding sites on the protein, and, depending upon the characteristics of the resin, limited protein access to the immobilized metal ion. Thus, it is extremely difficult a priori to state which proteins will and which will not exhibit an affinity for immobilized metal ions.
Once binding has occurred, however, the protein can be released by protonation of its associated metal ion-binding ligand. Dissociation is achieved by lowering the pH of the surrounding buffer medium, a most common method for eluting bound proteins.
It has now been discovered that it is possible to apply the concept of immobilized metal ion affinity chromatography to the purification of a wide range of substances, and it is this discovery that forms the basis of the present invention. Thus, this invention is directed (1) to compounds specifically tailored so as to be readily purified via IMAC from mixtures containing such compounds and (2) to a process for such purification.